The present invention relates to a process for the preparation of thromboplastin from animal tissue, using an extraction with an aqueous salt solution, followed by separation of tissue material in order to obtain a thromboplastin containing salt solution.
An extraction process is known from European patent application EP-A-0 083 773. According to the known process, an acetone powder from the animal tissue, i.e. mammal tissue, particularly brain tissue, is initially prepared. The acetone powder is then extracted with an aqueous salt solution comprising 1-20 mM calcium ions and possibly a surface active substance.
Based on recent scientific research, it appears that using bovine brain tissue and bovine nerve tissue can lead to the transfer of the causative agents of BSE (Bovine Spongiform Encephalitis); (Spongiform Encephalopathy Advisory Committee, September 1994. Transmissible Spongiform Encephalopathies. A Summary of Present Knowledge and Research.xe2x80x9d HMSO, London, U.K.). Furthermore, it appears that, in order to prepare a satisfactory solution of thromboplastin, first preparing an acetone powder of the relevant tissue, and then carrying out the extraction with a solution of calcium salts and possibly a surface active substance is unnecessary.
The process according to the invention is characterized in that muscular tissue, obtained from mammals or from fish, is directly extracted with an aqueous salt solution.
The process according to the present invention is characterised in that muscular tissue, obtained from mammals or from fish, is directly extracted with an aqueous salt solution.
Smariga, P. E. and Maynard, J. R. (1982) Blood, 60, 140-147 relates to the effects of platelets on the tissue factor and the fibrinolytic inhibition of cultured human fibroblasts and vascular cells. From Derwent Abstract AN 92-388343-XP002089073 relating to SU 88 616 530 A, a process for the preparation of thromboplastin from brain tissue-containing pieces of tissue from 24 hours of chickens is known. Biological Abstracts (1997) 1997, no. 107757 relates to the presence of thromboplastin activity in human gastric tissues.
By using the present process, the BSE problem can be avoided. Moreover, a more simple and less laborious preparation method is obtained, since, according to the invention, the step of preparing an aceton powder is unnecessary. The muscular tissue is simply reduced to smaller pieces and is then extracted. A salt solution without calcium ions or a surface active substance is sufficient.
The muscular tissue which is used is preferably derived from mammals, especially from bovine species, as said muscular tissue can be obtained in large amounts. The process can also be applied advantageously to muscular tissue from fish.
It was experimentally demonstrated that the used muscular tissue is preferably not derived from a freshly slaughtered animal, but that it has an xe2x80x9cagexe2x80x9d of 4-7 days. Extracts from such an xe2x80x9cagedxe2x80x9d muscular tissue have a higher thromboplastin activity. A higher thromboplastin activity is also obtained with vacuum packed muscular tissue with an age of 2 weeks.
Concerning the composition of the aqueous salt solution to be used for extracting, it is preferred that it is buffered to a pH value of 6.0-7.5. Using a phosphate salt is particularly preferable, as said salt also has a good buffering action.
During the preparation according to the invention a temperature of 0-4xc2x0 C. is maintained and principle. However, a higher yield of thromboplastin activity is obtained if the extraction is carried out with salt solution at a temperature of 40-50xc2x0 C.
The separation of the tissue material following the extraction is preferably carried out by centrifugation. Other separation methods lead to losses and/or are more time-consuming. Thus, filtration leads to losses by adsorption of, for instance, phospholipids, if a paper filter is used, and to higher costs if synthetic filters with a special composition and a particular molecular weight threshold is used in pressurized filtration devices.
The thromboplastin obtained by using the process according to the present invention can for instance be used for nutritional purposes. For such an application, the extraction has to be carried out with a solution of salts which are authorised for nutritional products, such as cooking salt, phosphates and acetates. Concerning an application in meat products, the thromboplastin is of course preferably derived from the same animal species as the species of the meat itself.
The present invention is further illustrated by examples.